Modulation of Oxidative phosphorylation by mitochondrial soluble adenylyl cyclase

描述（由申请人提供）：线粒体蛋白磷酸化是一种快速有效的调节氧化磷酸化（OXPHOS）和维持能量稳态的方法。线粒体PKA通过蛋白质磷酸化调节电子传递链上的酶，这一过程受信号通路调控，包括线粒体可溶性腺苷酸环化酶（sAC），产生cAMP，激活PKA。这种信号级联作为代谢传感器，调节线粒体中的能量转换。细胞色素氧化酶（COX）是ETC通量的起搏器，其活性受其亚基磷酸化和ATP变构抑制的调节。我们发现COX是信号通路的靶标，并鉴定出COX的亚基IV （COXIV）是磷酸化的靶标。我们还证明了COXIV- 1中S58的磷酸化负责调节COX活性。我们认为这一调控途径参与了对OXPHOS缺陷的代谢适应性反应，并可能成为药物干预的靶点。本项目的目的是了解体内线粒体内sAC-cAMP- PKA信号和S58 COXIV-1磷酸化的生理意义。我们将了解这个系统在健康组织和受线粒体缺陷影响的组织中的行为。为此，我们将培育和研究小鼠，对调控途径的分子参与者进行基因改造，并评估临床表型和线粒体生物化学的结果。在目标1中，我们将产生转基因小鼠，表达选择性靶向线粒体基质（mito-sAC）的可诱导sAC，有望产生高基础代谢。在ai2中，我们将产生缺乏磷酸化位点的COXIV-1 S58A敲入小鼠，因此无法上调ETC通量和增强ATP产生，导致运动不耐受，葡萄糖利用率降低，产热功能受损，脂肪储存增加。在目的3中，我们将研究由条件和诱导性COX基因破坏引起的OXPHOS缺陷小鼠模型中sAC-cAMP-PKA的调节。这些小鼠概括了线粒体疾病的生化和临床特征。我们将评估受影响组织中sAC-cAMP-PKA调控和线粒体蛋白磷酸化的机制，并将其与疾病进展联系起来。

DESCRIPTION (provided by applicant): Phosphorylation of mitochondrial proteins is a rapid and efficient way to regulate oxidative phosphorylation (OXPHOS) and maintain energy homeostasis. Mitochondrial PKA modulates enzymes of the electron transfer chain through protein phosphorylation, which is regulated by a signaling pathway, involving mitochondrial soluble adenylyl cyclase (sAC), generating cAMP that activates PKA. This signaling cascade serves as a metabolic sensor that modulates energy conversion in mitochondria. Cytochrome oxidase (COX) is a pacemaker of ETC fluxes, whose activity is modulated by phosphorylation of its subunits and by ATP allosteric inhibition. We showed that COX is a target of the signaling pathway and identified subunit IV of COX (COXIV) as a target for phosphorylation. We also demonstrated that phosphorylation of S58 in COXIV- 1 is responsible for modulation of COX activity. We propose that this regulatory pathway participates to metabolic adaptive responses to OXPHOS defects and could be a target for pharmacological intervention. The goal of this project is to understand the physiological implications of intramitochondrial sAC-cAMP- PKA signaling and S58 COXIV-1 phosphorylation, in vivo. We will understand how this system behaves in healthy tissues and in tissues affected by mitochondrial defects. To this end, we will generate and study mice, in which the molecular players of the regulatory pathway are genetically modified and assess the outcomes on clinical phenotype and mitochondrial biochemistry. In aim 1, we will generate transgenic mice expressing inducible sAC selectively targeted to the mitochondrial matrix (mito-sAC), which is expected produce high basal metabolism. In aim2, we will generate COXIV-1 S58A knockin mice, lacking the phosphorylated site, and thus incapable of up-regulating ETC fluxes and enhancing ATP production, resulting in exercise intolerance, decreased glucose utilization, impaired thermogenesis, and increased fat storage. In aim 3, we will investigate sAC-cAMP-PKA modulation in a mouse model of OXPHOS defect caused by conditional and inducible genetic disruption of COX. These mice recapitulate biochemical and clinical characteristics of mitochondrial diseases. We will assess how sAC-cAMP-PKA modulation and protein phosphorylation in mitochondria from affected tissues and correlate it with disease progression.